Protocol to quantify bacterial burden in time-kill assays using colony-forming units and most probable number readouts for Mycobacterium tuberculosis

• Published in: STAR protocols Vol. 6; no. 1; p. 103643
• Main Authors: Rabodoarivelo, Marie Sylvianne, Hoffmann, Eik, Gaudin, Cyril, Aguilar-Ayala, Diana Angélica, Galizia, Jordana, Sonnenkalb, Lindsay, Dal Molin, Michael, Cioetto-Mazzabò, Laura, Degiacomi, Giulia, Recchia, Deborah, Rybniker, Jan, Manganelli, Riccardo, Pasca, Maria Rosalia, Ramón-García, Santiago, Lucía, Ainhoa
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.03.2025; Elsevier
• Subjects: Bacterial Load - methods; Cell Biology; Clinical Protocol; Colony Count, Microbial - methods; Colony-Forming Units Assay - methods; High Throughput Screening; Humans; Microbial Sensitivity Tests - methods; Microbiology; Molecular Biology; Mycobacterium tuberculosis - drug effects; Mycobacterium tuberculosis - growth & development; Protocol; High Throughput Screening; Microbiology; Molecular Biology; Clinical Protocol; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2025.103643

Here, we present a protocol to perform a time-kill assay (TKA) to quantify bacterial burden at multiple time points using colony-forming units and most probable number readouts simultaneously. We describe steps for preparing inoculum, experimental conditions, and sampling bacterial counts. We then detail procedures for quantification and analysis. TKAs provide longitudinal data reflecting the dynamics of the antibiotic effect over time against a planktonic culture and quantify the concentration-effect relationship. For complete details on the use and execution of this protocol, please refer to Van Wijk et al.1 [Display omitted] •The protocol describes inoculum preparation and conditions for time-kill assays•MPN in a 96-well plate and 2.5 μL CFU plating techniques used as simultaneous readouts•Detailed procedures for quantifying and analyzing MPN and CFU data Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol to perform a time-kill assay (TKA) to quantify bacterial burden at multiple time points using colony-forming units and most probable number readouts simultaneously. We describe steps for preparing inoculum, experimental conditions, and sampling bacterial counts. We then detail procedures for quantification and analysis. TKAs provide longitudinal data reflecting the dynamics of the antibiotic effect over time against a planktonic culture and quantify the concentration-effect relationship.